Different Mechanisms Preserve Translation of Programmed
Cell Death 8 and JunB in Virus-Infected Endothelial Cells
Huimiao Jiang, Hansjörg Schwertz, Douglas I. Schmid, Brandt B. Jones, John Kriesel,
Mark L. Martinez, Andrew S. Weyrich, Guy A. Zimmerman, Larry W. Kraiss
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Objective—Translation initiation of eukaryotic mRNAs typically occurs by cap-dependent ribosome scanning mechanism.
However, certain mRNAs are translated by ribosome assembly at internal ribosome entry sites (IRESs). Whether
IRES-mediated translation occurs in stressed primary human endothelial cells (ECs) is unknown.
Methods and Results—We performed microarray analysis of polyribosomal mRNA from ECs to identify IRES-containing
mRNAs. Cap-dependent translation was disabled by poliovirus (PV) infection and confirmed by loss of polysome peaks,
detection of eukaryotic initiation factor (eIF) 4G cleavage, and decreased protein synthesis. We found that 87.4% of
mRNAs were dissociated from polysomes in virus-infected ECs. Twelve percent of mRNAs remained associated with
polysomes, and 0.6% were enriched ⱖ2-fold in polysome fractions from infected ECs. Quantitative reverse
transcription–polymerase chain reaction confirmed the microarray findings for 31 selected mRNAs. We found that
enriched polysome associations of programmed cell death 8 (PDCD8) and JunB mRNA resulted in increased protein
expression in PV-infected ECs. The presence of IRESs in the 5= untranslated region of PDCD8 mRNA, but not of JunB
mRNA, was confirmed by dicistronic analysis.
Conclusion—We show that microarray profiling of polyribosomal mRNA transcripts from PV-infected ECs successfully
identifies mRNAs whose translation is preserved in the face of stress-induced, near complete cessation of cap-dependent
initiation. Nevertheless, internal ribosome entry is not the only mechanism responsible for this privileged translation.
(Arterioscler Thromb Vasc Biol. 2012;32:997-1004.)
Key Words: IRES 䡲 JunB 䡲 PDCD8 䡲 microarray 䡲 poliovirus
T
ranslation initiation is the major regulatory checkpoint of
eukaryotic gene expression.1,2 Two distinct mechanisms
of translation initiation, cap-dependent and cap-independent,
are recognized.
Cap-dependent initiation, also known as the scanning
model, is the mechanism by which most eukaryotic mRNAs
are translated. All nuclear encoded eukaryotic mRNA is
processed by capping with 7-methylguanosine at the 5=terminus before transport out of the nucleus and into the
cytoplasm for translation. In the cytoplasm, the
7-methylguanosine cap structure is bound by eukaryotic
initiation factor (eIF) 4F, a complex of 3 polypeptides
including eIF4E, A, and G. The 7-methylguanosine cap
structure is recognized by eIF4E which serves to anchor the
eIF4F complex, whereas the secondary structure of the
mRNA is unwound by the helicase activity of eIF4A. eIF4G
functions as a scaffolding protein that bridges the mRNAbound eIF4F to the 40S ribosomal subunit through eIF3. This
preinitiation complex then scans the 5= untranslated region
(UTR) until an AUG start codon is recognized.3
An alternative, and less common, form of translation
initiation is a cap-independent mechanism that requires an
internal ribosome entry site (IRES), a specialized internal
RNA structure in the 5=UTR. During cap-independent translation, 40S ribosomal subunits assemble directly on the IRES
near the start codon, thereby bypassing the requirement for
the mRNA 5=cap structure and eIF4F to initiate translation.4
First identified in the 5=UTRs of mRNAs of encephalomyocarditis virus and poliovirus (PV),5,6 IRESs allow the
efficient translation of uncapped viral messages into proteins
while cap-dependent translation initiation is inhibited in the
host cell. Since their identification, a number of cellular
mRNAs have been recognized to contain an IRES.4 These
IRES-containing mRNAs encode proteins involved in multiple biological processes, such as mitosis, differentiation,
apoptosis, hypoxia, heat shock, and oxidant injury.7 IRESs
maintain or even induce the synthesis of specific proteins,
whereas cap-dependent translation is severely impaired in
cells under stress conditions. Dysregulation of IRESmediated translation has been linked to the development of
Received on: July 6, 2011; final version accepted on: January 20, 2012.
From the Division of Vascular Surgery (H.S., L.W.K.), Department of Internal Medicine (B.B.J., J.K., A.S.W., G.A.Z.) and the Program in Molecular
Medicine (H.J., H.S., D.I.S., M.L.M., A.S.W., G.A.Z., L.W.K.), University of Utah, Salt Lake City, UT.
The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/doi:10.1161/ATVBAHA.111.245324/-/
DC1.
Correspondence to Larry W. Kraiss, MD, Division of Vascular Surgery, University of Utah Health Sciences Center, Room 3C344, 30 North 1900 East,
Salt Lake City, UT 84132. E-mail larry.kraiss@hsc.utah.edu
© 2012 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org
997
DOI: 10.1161/ATVBAHA.112.245324
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several human diseases, including diabetes,8 cardiovascular diseases,9 multiple myeloma,10 Charcot-Marie-Tooth disease,11 and congenital X-linked dyskeratosis.12 Therefore,
delineation of conditions in which IRESs influence patterns
of protein expression has substantial biological and pathological significance.
Endothelial cells (ECs), which line the extensive human
vasculature, respond to a multitude of signals from their
environment and directly influence complex homeostatic
mechanisms that regulate vessel injury, vascular tone, inflammation, and coagulation. ECs are capable of both rapid and
prolonged responses to extracellular stimuli, resulting in
alterations in cellular function and phenotype. Pathological
conditions such as sepsis, ischemia-reperfusion injury, and
vascular thrombosis may arise in part because of dysregulated
endothelial responses to these stimuli. Most studies in ECs
have focused on transcriptional mechanisms that regulate
gene expression, which may require hours for a response to a
stimulus because new mRNA production is a requisite event.
We and others have shown that ECs can also regulate gene
expression by translational control as a means of responding
to stimuli in relatively short time periods and with diversity
and precision that is not afforded by transcriptional regulation
alone.2,13–18 Whether ECs use IRESs as a translational control
mechanism when cap-dependent translation is inhibited by
cellular stress is unknown. We speculated that human ECs are
capable of IRES-mediated protein synthesis and that this
mechanism might be used when ECs are activated under
pathophysiologic conditions or subjected to pathological
stress.
To approach this question, we established PV infection of
cultured human ECs as a model for internal ribosome entry
when cap-dependent translation is inhibited in stressed vascular endothelium. We then performed polyribosomal (polysome) profiling and high-throughput microarray analysis19
and identified several IRES-containing mRNAs in ECs infected with PV. Programmed cell death 8 (PDCD8), JunB,
and angiomotin-like 2 (AMOTL2) emerged as intriguing
candidates for IRES-mediated translation as the presence of
an IRES has not been described in the mRNAs encoded by
these genes in any cell type. We show that ECs use capindependent translation as an important alternative mechanism to selectively synthesize gene products that may be
necessary for the cell’s response to stress when capdependent translation is inhibited.
Methods
Additional assays were performed according to standard techniques:
immunofluorescence; immunoblot analysis; polysome profiling;
[35S]methionine incorporation experiment; quantitative real-time
reverse transcription–polymerase chain reaction (qRT-PCR); construction of dicistronic vectors; transient transfections, and luciferase
reporter assays; and Northern analysis. An expanded Methods
section is available in the online-only Data Supplement.
EC Culture and Virus Preparation
Primary human umbilical vein ECs were isolated and cultured as
described.20 EaHy 926 cells have been propagated in our laboratory
since the original gift from Edgell.21 PV1 (live-attenuated vaccine
strain) was purchased from American Type Culture Collection. The
Figure 1. Poliovirus (PV) infects human endothelial cells (ECs).
ECs grown on 8-well glass chamber slides were incubated with
VERO cell medium in the absence of PV1 (mock) or infected
with PV1 for the indicated times. Cells were fixed and stained
for PV1 antigen (green) and nuclear DNA (blue) as described in
the online-only Data Supplement (n⫽3).
preparation of PV1 and its infection of ECs are described in the
online-only Data Supplement.
Microarray Hybridization
RNA from polysome fractions was isolated from mock or PVinfected ECs using Trizol LS (Invitrogen, Carlsbad, CA), labeled
with Cy3 (mock-infected) or Cy5 (PV-infected), and then subjected
to whole human genome-wide microarray analysis (43 203 transcripts, Agilent, Santa Clara, CA) to obtain translational profiles. The
procedures were performed in the microarray core facility in University of Utah as described in the online-only Data Supplement.
Microarray Data Analysis
Each Cy3 or Cy5 signal was normalized by total Cy3 or Cy5 signal
on the slide respectively. The ratios of normalized Cy5 to Cy3 were
then calculated. We arbitrarily used PV-infected/mock-infected ratio
thresholds of ⱖ2.0 as indicating translational upregulation, 1.0 to 2.0
as preserved translation and ⱕ1.0 as disrupted translation in response
to PV infection.
Results
Verification of Disruption of Cap-Dependent
Translation in PV-Infected ECs
PV disrupts host cap-dependent translation while using
IRESs to translate its own viral message.6 Host messages that
continue to be translated despite PV infection are also likely
to do so via an IRES-dependent mechanism.19 To determine
whether PV infection can be used to identify IRES candidates
in human ECs, we first investigated the potential for PV to
infect primary ECs in culture. Using immunocytochemistry,
we demonstrated the presence of PV antigen in the cytoplasm
of ECs as early as 2 hours postinfection (Figure 1). Between
4 and 6 hours after inoculation, PV protein dramatically
increased in the cytoplasm of most cells, indicating active and
efficient translation of PV mRNA into protein during this
time period. By 8 hours postinfection, EC lysis was apparent,
suggesting that the PV had completed its first life cycle.22
PV inhibits translation of host cellular mRNAs by proteolytic cleavage of eIF4G, a key component of cap-dependent
translation initiation machinery.19 There are 2 isoforms of
eIF4G in eukaryotic cells, I and II, both of which need to be
depleted to completely inhibit host cap-dependent transla-
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Figure 2. Poliovirus (PV) infections disrupt
cap-dependent translation in human
endothelial cells (ECs). A, eukaryotic initiation factor (eIF) 4GI (left panel) and
eIF4GII (right panel) were cleaved in
PV-infected ECs. Cell lysates from
500 000 mock- and PV-infected ECs were
separated by SDS-PAGE and immunoblotted with antibodies directed against
eIF4GI or eIF4GII (n⫽5). B, Loss of
polyribosome peaks in profiles from
PV-infected ECs. Cytoplasmic lysates
from mock- and PV-infected ECs were
fractionated by sucrose density gradient
centrifugation. The absorbance profile at
254 nm is shown, and the position of
40S, 60S, and 80S ribosomal subunits
and polysomes are indicated (n⫽5). C,
Inhibition of protein synthesis in
PV-infected ECs. New protein synthesis in
both mock- and PV-infected (4 hours)
ECs was measured by [35S]methionine
incorporation. Radiolabeled protein products in cell lysates were separated by
SDS-PAGE. An autoradiography of the gel
is shown. Visible bands in the PV lane
likely represent newly synthesized viral
protein.42
tion.23,24 To determine whether eIF4G cleavage results from
PV infection in human ECs, Western analysis for eIF4G
isoforms was performed. We found cleavage of both isoforms
beginning 2 hours after infection. After 4 hours of PV
infection, eIF4GI was completely cleaved along with the
majority of eIF4GII (Figure 2A). Thus, a 4-hour time point
was chosen to use in later microarray experiments in which
we examined candidate IRES-containing messages in virally
infected ECs. Interestingly, complete cleavage of eIF4GII
was never observed in ECs infected with PV even 6 hours
postinoculation.
Further evidence for inhibition of host translation by PV
was provided by polyribosomal profiling of PV-infected ECs.
In mock-infected ECs, multiple polysome peaks were observed, consistent with efficient translation of a multitude of
host EC proteins1,2 (Figure 2B, left). Four hours after PV
infection, the polysome peaks were dramatically reduced in
parallel with a corresponding increase in the abundance of the
40S, 60S, and 80S fractions (Figure 2B, right). These changes
in the polyribosomal profiling pattern of PV-infected ECs are
consistent with major inhibition of cap-dependent translation
at the initiation stage in the host cell.19,25,26
To further assess whether the loss of polysome peaks is an
indicator of global inhibition of protein production in PVinfected ECs, labeling of new protein products using [35S]methionine incorporation was performed. As expected, there
was a dramatic inhibition of protein synthesis in ECs 4 hours
postinfection (Figure 2C). Nevertheless, several protein
bands were readily detected, providing evidence for synthesis
of viral proteins and, potentially, a subset of host proteins via
alternative mechanisms when cap-dependent translation is
disabled by the infecting virus (Figure 2C).
Identification of Novel IRES-Containing mRNAs
in Virus-Infected ECs
The most likely mechanism of preserved synthesis of host
proteins in PV-infected cells is IRES-mediated translation.19
To examine this issue, candidate genes were first identified in
ECs by comparative genome-wide microarray profiling of
mRNAs that remained associated with polysomes (sucrose
gradient fractions 6 –10, with 3 or more associated ribosomes
per mRNA) following PV infection (Figures 2B and 3A). For
each array element, the amount of polysome-associated
mRNA derived from PV-infected ECs was compared with the
amount from mock-infected ECs to obtain polysome ratios
(PR). PR values greater than 2.0 were taken to indicate
increased association with polysomes in PV-infected cells,
and the mRNA was considered a strong candidate for the
presence of an IRES (Figure 3A).
As expected in the polysome arrays from PV-infected ECs,
the majority (37 756 of 43 203, 87.4%) of host mRNAs
exhibited a PR ⬍1 (Figure 3B), indicating reduced or absent
association with polysomes when cap-dependant translation
was inhibited. An example is the housekeeping gene ␤-actin
(ACTB), with a PR of 0.69⫾0.08. In contrast to the large
number of genes that had reduced association with polysomes, 12% of mRNAs remained associated with polysomes
(1⬍PR⬍2) from ECs infected by PV. Interestingly, 277 of
43 203 (0.64%) mRNAs showed 2-fold or more enrichment in
the polysome fractions from PV-infected ECs compared with
mock-infected cells (Figure 3B). These 277 messages represented 241 unique genes and encoded proteins involved in
multiple biological functions, including: angiogenesis, cell
signaling, growth and apoptosis, oncogenesis, and inflammation (Table I in the online-only Data Supplement). Some of
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Figure 4. Quantitative real-time reverse transcription–polymerase chain reaction (qRT-PCR) analysis of selected transcripts
confirmed polysome ratio calculations from microarray analysis.
Scatter plot comparing PR determined by microarray vs qRTPCR. Particular sequences of interest are indicated. The full set
of mRNA sequences tested for correlation is reported in Table III
in the online-only Data Supplement.
Figure 3. Identification of candidate internal ribosome entry site
(IRES)– containing mRNAs in poliovirus (PV)–infected endothelial
cells (ECs) using microarray. A, Microarray approach for the
analysis of polysomes from mock- and PV-infected ECs. Fractions from sucrose density gradient analysis were pooled into
subpolysomes (fractions 1–5) and polysomes (fractions 6 –10)
Polysome-associated mRNAs isolated from mock- and
PV-infected ECs were subjected to a genome-wide microarray
analysis. Polysome ratios (PRs) (Cy5:Cy3) were obtained by
comparing signals from PV-infected cells to those from mockinfected ECs. A PR value greater than 2.0 suggested polysome
association and persistent translation in PV-infected cells. B,
Polysome microarray data summary. A very small subset of
transcripts with a PR ⬎2 was identified. Approximately 12% of
transcripts from PV-infected ECs retained some association with
polyribosomes (PR⫽1–2).
these messages have previously been reported to contain
IRESs, including Cyr61 and Pim1.19 (The microarray data
have been deposited in NCBI’s Gene Expression Omnibus
(http://www.ncbi.nlm.nih.gov/geo/) and are accessible
through Gene Expression Omnibus Series accession number
GSE15356.)
Confirmation of Microarray Predictions
Using qRT-PCR
To verify the microarray data, 31 genes with a wide range of
PR values derived from microarray analysis were chosen, and
PCR primers were designed accordingly (Table II in the
online-only Data Supplement). A PR value for each sequence
was generated by using qRT-PCR to compare the relative
abundance of mRNA isolated from the polysome fractions of
PV-infected ECs to mock-infected ECs. Table III in the
online-only Data Supplement shows PR values generated
from both the array and qRT-PCR for these 31 genes.
Overall, PR generated from qRT-PCR were highly similar to
those obtained from microarray experiments with a correlation coefficient of 0.92 (Figure 4). Specifically, the housekeeping gene ␤-actin had a PR of 0.69⫾0.08 from array
analysis, which was confirmed by qRT-PCR with a calculated
PR 0.46⫾0.06 (Table III in the online-only Data Supplement). These data indicate that ␤-actin mRNA is dissociated
from polysomes following PV infection. Conversely, some
messages increased their association with polysomes following inhibition of cap-dependent translation by PV. For example, PDCD8 and AMOTL2 had a PR of 2.4 and 3.0 from array
analysis and 2.1⫾1.0 and 1.8⫾0.6 from qRT-PCR, respectively (Table III in the online-only Data Supplement). Although PR of JunB from qRT-PCR (0.9) was less than that
from array analysis (2⫾0.8), the data still indicated its
retained association with polysomes in PV-infected ECs.
Thus, JunB was still considered a candidate to contain an
IRES in its 5=UTR.
PDCD8 and JunB Protein Expression Is Increased
in PV-Infected ECs
To determine whether preserved or increased association of
mRNAs with polyribosomes in PV-infected ECs is also
associated with translation of those transcripts and synthesis
of the corresponding proteins, immunodetection assays were
performed. Immunocytochemistry was used to examine accumulation of PDCD8 in PV-infected ECs. Mock-infected
ECs were found to have basal levels of PDCD8 protein
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Figure 5. PDCD8 and JunB protein expression is increased in
poliovirus (PV)–infected endothelial cells (ECs). A, ECs grown on
8-well glass chamber slides were infected with PV1 for 6 hours,
fixed, and stained for PV1 antigen (green), PDCD8 (red), and
nuclear DNA (nDNA) (blue) (n⫽2). B, ECs grown on 8-well glass
chamber slides were infected with PV1 for 6 hours, fixed, and
stained for PV1 antigen (green), JunB (red), and nDNA (blue)
(n⫽1). Ab indicates antibody. C, JunB protein was detected by
Western analysis of lysates from mock- and PV-infected ECs
using conditions similar to those in B (n⫽2).
diffusely distributed in the cytoplasm (Figure 5A). Following
6 hours of PV infection, increased PDCD8 protein was
observed throughout the cytoplasm of infected ECs, consistent with new translation of PDCD8 mRNA into protein.
Similarly, JunB protein increased in PV-infected ECs compared with mock-infected ECs as demonstrated by immunocytochemistry (Figure 5B) and Western blot analysis (Figure
5C). AMOTL2 protein expression was not examined in these
experiments because no appropriate anti-AMOTL2 antibody
was available.
Functional Analysis of IRESs in Candidate
mRNAs Using Dicistronic Reporter Assays
No unequivocal consensus sequences for IRESs have been
defined.27 One approach to identify mRNAs with a functional
1001
Figure 6. A dicistronic assay indicates that internal ribosome
entry site (IRES) activity is present in the PDCD8 5= untranslated
region (UTR). A, Constructs used in this analysis are schematically illustrated. luc indicates luciferase. B, EaHy cells were
transfected with control and these reporter constructs. Ratios of
fyrefly luciferase (fLUC) to renilla luciferase (rLUC) activity for
constructs illustrated in A are shown. The ratios of luciferase
activities obtained from transfection experiments with the control empty vector plasmid were set to 1. Assays were performed
in duplicate, and results represent the average of 3 independent
experiments. C, Northern analysis was used to detect the presence of monocistronic constructs. Poly(A) mRNA was isolated
from EaHy cells transfected with dicistronic plasmids and analyzed by Northern blot hybridization using radiolabeled RNA
probes complementary to fLUC sequences. The migration of
28S and 18S rRNA is indicated.
IRES sequence in the 5=UTR is the use of a dicistronic assay
in which expression of a reporter driven by IRES activity is
compared with expression of a second reporter upstream of
the candidate IRES sequence.28 To examine PDCD8, JunB,
and AMOTL2 mRNAs for functional IRES elements, the
5=-UTR sequences from these genes were positioned in the
intercistronic space between renilla luciferase (rLUC) and
fyrefly luciferase (fLUC) cistrons. These constructs were then
transfected into EaHy 926 cells, a human EC line,21 and
expression levels were determined. In this assay, the translation of upstream rLUC is cap-dependent, whereas the translation of downstream fLUC depends on the intercistronic
5=UTR containing an IRES (Figure 6A).28 An empty dicistronic vector and a dicistronic plasmid containing the ␤-actin
5=UTR were used as controls. The dicistronic data are
displayed as the ratio of fLUC to rLUC activity with the ratio
obtained from the empty dicistronic vector set to 1. Constructs containing the PDCD8 and AMOTL2 5=UTRs yielded
fLUC/rLUC ratios of 10.5 and 125.7, respectively, whereas
the constructs containing the actin 5=UTR only increased
fLUC/rLUC ratio 3.8-fold compared with the empty vector
(Figure 6B). These data suggested that the PDCD8 and
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AMOTL2 5=UTRs contained IRES activities. Interestingly,
inserting the JunB 5=UTR into the intercistronic region only
increased the fLUC/rLUC activity ratio by 5.8-fold (approximately the same level as the actin 5=UTR, Figure 6B). This
result suggests that preserved translation of JunB mRNA in
PV-infected cells did not require an IRES.
Apart from the presence of an IRES, alternative explanations for increased synthesis of the downstream fLUC reporter product in the dicistronic assay are cryptic transcriptional promoter activity in the cloned 5=UTR or alternative
splicing introduced by the candidate 5=UTR.27 To exclude
these possibilities, Northern analysis was performed using
polyA mRNA isolated from the transfected EaHy cells to
determine whether monocistronic fLUC mRNA was produced from these constructs, thus suggesting unexpected
promoter activity. The size of the Northern product would
also indicate whether alternative splicing had occurred.
Northern blots revealed that intact dicistronic transcripts were
present in the RNA samples isolated from cells transfected
with empty vector, actin, PDCD8, and AMOTL2 dicistronic
constructs (Figure 6C). However, in cells transfected with the
AMOTL2 dicistronic construct, an equal amount of monocistronic intact fLUC mRNA was found. Thus, monocistronic
transcripts likely contributed to the fLUC activity observed in
the AMOTL2 dicistronic transfection probably explaining the
particularly dramatic increase in downstream fLUC synthesis
(126-fold) seen with this construct. Because of this artifact,
we are unable to conclusively determine whether AMOTL2
contains an IRES.
Discussion
ECs respond to inflammation, injury, and stress signals by
undergoing key changes in function and phenotype, many of
which require new or altered gene expression.29 Survival and
function of mammalian cells exposed to environmental and
toxic stress requires reprogramming of mRNA translation to
sustain expression of key gene products,30 but these intricate
processes are largely unexplored in human endothelium. Our
studies provide new insights regarding human ECs and their
translational responses under conditions of experimental
stress imposed by viral infection.
Use of Polysome Profiling and High-Throughput
Microarray Analysis to Identify Privileged
Translation of mRNAs During Cell Stress
The combination of polysome profiling and microarray analysis has been used to identify candidate IRES-containing
messages in several other cell systems using different mechanisms of cellular stress.19,25,26 Approximately 3% to 5% of
cellular mRNAs were found to be translated using capindependent initiation under each condition tested. In these 3
studies, there was no significant overlap among the genes
identified, suggesting that up to 10% to 15% of all mRNAs
are capable of using cap-independent translation initiation
mechanisms.31 In our EC PV infection model, we found that
approximately 12% of EC mRNAs remain associated with
the polysomes. This relatively higher percentage of candidate
mRNA molecules in our system could be due to the experimental procedures used to pool polysome fractions, different
cell types, virus strain, stress stimulators, microarray procedures, and materials used in the studies.
Because complete cleavage of eIF4GII was never observed
in PV infected ECs, we cannot exclude the possibility that a
very small amount of cap-dependent initiation persisted.
Nonetheless, these studies indicate that translation of up to
15% of mRNA transcripts may be preserved under severe,
ultimately lethal, cell stress. Adaptive mechanisms to allow
ongoing translation initiation of privileged transcripts represent an underappreciated pathway to gene expression in
stressed human ECs.
PDCD8 Function in PV-Infected Cells
PV is the causative agent of poliomyelitis, in which motor
neuron death leads to paralysis. PV-induced motor neuron
death was recently found to be mediated through an apoptotic
process.32–34 PV was also reported to induce apoptosis in
vitro in other cell types, including the CaCo-2 colon cancer
cell line,35 the U937 promonocytic cell line,36 dendritic cells
and macrophages,37 and HeLa cells.38,39 However, the mechanisms by which PV induces apoptosis are not totally clear.
Induction of PV2A protease has been reported to result in
caspase-independent apoptotic cell death.40 The proposed
mechanism was PV-induced preferential cap-independent
translation of cellular mRNAs that encode apoptotic factors.
This hypothesis is supported by the finding that several
IRES-containing mRNAs encoded proteins regulate apoptosis.41–44 A recent article reported that approximately 3% of
mRNAs remain associated with the polysomes in apoptotic
cells.25 In this report, we identified another apoptotic factor,
PDCD8, that contains an IRES element in its 5=UTR.
PDCD8, also called apoptosis-inducing factor, has been
reported to mediate caspase-independent human coronary EC
apoptosis induced by oxidized low-density lipoprotein.45 In
our PV-infected EC model, we found that PDCD8 mRNAs
were preferentially associated with heavy polysomes and
efficiently translated during PV infection. Ongoing PDCD8
synthesis in infected cells is possibly a host defense mechanism to induce apoptosis in virally infected cells.
Translation of JunB and Other Activator Protein
1 Members in PV-Infected ECs
Once infected by virus, host cells initiate an antiviral defense
response. This includes the increased expression of
immediate-early genes, including the activator protein 1
transcription factor family, which affect cell survival and the
outcome of the viral infection.46 JunB, a member of the
activator protein 1 family, is translationally upregulated in
thrombin-stimulated ECs (Schmid et al, manuscript in preparation, 2011)47 and is induced on virus infection.46
Using microarray and qRT-PCR, we demonstrated that
JunB message remains associated with polysomes in PVinfected ECs. Moreover, the persistent polysome association
of JunB mRNA correlates with increased JunB protein
expression in PV-infected cells. However, the dicistronic
assay did not confirm the presence of an IRES in the JunB
5=UTR because the fLUC/rLUC activity ratio approximated
that of the actin 5=UTR, a transcript that dissociates from
polysomes in virus-infected ECs. This result indicates that
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persistent translation of JunB mRNA in PV-infected cells
may not require an IRES and is consistent with previous
reports that JunB 5=UTR does not contain an IRES.42,48
Despite the lack of evidence for an IRES in the 5=UTR of
JunB, evidence for its preserved translation in the face of PV
infection is compelling. Both microarray and qRT-PCR
analysis demonstrated that JunB message remains associated
with polysomes in PV-infected ECs. Moreover, the persistent
polysome association of JunB mRNA correlates with increased JunB protein expression in PV-infected cells as
shown by both Western blotting and immunocytochemistry.
We speculate that increased JunB protein expression in
PV-infected ECs occurs as a result of particularly avid
affinity of the JunB 5=UTR to surviving components of the
cap complex, possibly eIF4GII because this key protein was
incompletely proteolyzed (Figure 1B). We also found that
total JunB mRNA levels increased 1.8-fold on PV infection in
ECs (data not shown), which would increase the number of
JunB transcripts available to interact with eIF4GII. Interestingly, our microarray data showed that other activator protein
1 members FosB, c-Fos, and JunD exhibit PRs of 3.7, 2.1,
and 1.1 respectively, consistent with this gene family’s
biological role in cell stress.47 Thus, our studies with JunB in
PV-infected ECs suggest that a privileged population of
cellular mRNA exist that are able to complete translation
initiation under stressful conditions, possibly by maintaining
particularly avid affinity to the cap complex.
Limitation of Dicistronic Assay Preclude
Identification of an IRES in AMOTL2
AMOTL2 belongs to the motin family, which is made up of 3
polypeptides: angiomotin, angiomotin-like 1 (AMOTL1), and
AMOTL2.49 Angiomotin binds to angiostatin and regulates
angiogenesis,50 and AMOTL 2 is essential for cell movement
in vertebrate embryos.51 Our microarray data imply that the
motin family mRNAs are subjected to differential translational regulation in ECs during PV infection. Angiomotin and
AMOTL1 exhibit a PR of 0.9⫾0.3 and 1⫾0.3, respectively,
whereas AMOTL2 has a PR of 3. qRT-PCR confirmed the
increased polysome association of AMOTL2 mRNA in ECs
during PV infection, again suggesting that AMOTL2 might
contain an IRES.
The dicistronic assay has been considered “a gold standard
for detecting IRES activity,”27 but it has limitations.52 Following performance of the assay, Northern blot analysis is
necessary to exclude several possible artifacts. Insertion of
the 5=UTR of interest into the intercistronic region might
unintentionally introduce a cryptic transcriptional promoter
for the downstream cistron. Alternative splicing might excise
the intercistronic segment and allow the 2 cistrons to be
translated as 1. Our dicistronic assay did show that the
AMOTL2 5=UTR dramatically increased the fLUC/rLUC
ratio compared with the empty dicistronic plasmid and
compared with the activity of the actin 5=UTR. However,
Northern analysis revealed abundant monocistronic fLUC
message along with intact dicistronic transcripts in cells
transfected with the rLUC-AMOTL2-fLUC reporter construct. It is likely that the monocistronic transcripts contributed to the fLUC activity observed in the AMOTL2 dicis-
1003
tronic assay clouding our ability to attribute the increased
fLUC expression to the presence of an IRES in the AMOTL2
5=UTR. Other experimental approaches, such as intact dicistronic mRNA transfection, will be needed to definitively
determine whether AMOTL2 5=UTR contains an IRES. Regardless, the notion that AMOTL2 might be preferentially
translated when ECs are stressed is consistent with the role of
angiogenesis as a reparative process.
We have conducted preliminary experiments (data not
shown) to assess whether similar mechanisms of translational
control are active in ECs subjected to nonviral stress, such as
hydrogen peroxide or arsenite. Oxidative stress produces an
overall decrease in protein synthesis and a shift of mRNA to
the monosomal fraction similar to the pattern depicted in
Figure 2B and 2C. Additionally, oxidative stress induces
expression of PDCD8 and JunB protein. These results suggest
that mechanisms to preserve translation of key stress-related
gene products are not unique to viral infection.
In summary, a model of PV infection of human ECs was
successfully established and produced evidence that up to
12% of cellular transcripts remain associated with the translational machinery, either through internal ribosome entry or
other mechanisms. These translational control mechanisms
further expand the diversity of regulated gene expression
displayed by ECs under severe stress.
Acknowledgments
Nahum Sonenberg and Elliot Spencer contributed important reagents, for which we are grateful. The University of Utah School of
Medicine Cell Imaging Facility was used to obtain confocal and
fluorescent images, and we greatly appreciate the aid of the core’s
director, Christopher K. Rodesch. We appreciate the aid of Donnell
Benson and Jessica Phibbs for cell culture and the significant
contributions of Diana Lim in preparing the figures for this article.
Sources of Funding
This work was supported by National Institutes of Health Grants
HL075507 (to L.W.K.), HL66277 (to A.S.W.), and R37HL44525 (to
G.Z.). Dr Schwertz was supported by a Beginning-Grant-in-Aid
(09BG1A 2250381) from the American Heart Association Western
States Affiliate.
Disclosures
None.
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Different Mechanisms Preserve Translation of Programmed Cell Death 8 and JunB in
Virus-Infected Endothelial Cells
Huimiao Jiang, Hansjörg Schwertz, Douglas I. Schmid, Brandt B. Jones, John Kriesel, Mark L.
Martinez, Andrew S. Weyrich, Guy A. Zimmerman and Larry W. Kraiss
Arterioscler Thromb Vasc Biol. 2012;32:997-1004; originally published online February 9,
2012;
doi: 10.1161/ATVBAHA.112.245324
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Online Data Supplements
for
Different Mechanisms Preserve Translation of Programmed Cell Death 8
(PDCD8) and JunB in Virus-Infected Endothelial Cells
Huimiao Jiang†, Hansjörg Schwertz*†, Douglas I. Schmid†, Brandt B Jones‡, John
Kriesel‡, Mark L.Martinez†‡, Andrew S. Weyrich†‡, Guy Zimmerman†‡, and Larry W.
Kraiss*†¶
Division of *Vascular Surgery, ‡Department of internal Medicine and the †Program in
Human Molecular Biology and Genetics, University of Utah, Salt Lake City, UT, 841125330
SUPPLEMENTARY MATERIALS AND METHODS
Reagents
PV type 1 (PV1) monoclonal antibody was from Chemicon International (United
Kingdom). Phospho-eIF4GI and PCDC8 antibodies were obtained from Cell Signaling
Technology (Danvers, MA). eIF4GII antibody was a gift from Dr. Nahum Sonenberg.
Cycloheximide was purchased from Sigma Chemical Co. (Saint Louis, MO). JunB
antibody was obtained from Active Motif (Carlsbad CA). Alexa-488 and Alexa-546conjugated secondary antibodies and TO-PRO-3 iodide were from Molecular Probes
(Eugene, OR).
1
Virus preparation
PV 1 (live-attenuated vaccine strain) was purchased from ATCC and amplified in
VERO cells in M199 supplemented with 20% fetal bovine serum. Media from VERO
cells cultured in parallel wells without adding PV were used for controls. The tissue
culture infective dose (TCID50) of the prepared PV stock was then determined in VERO
cells. EC were infected with PV1 stock at a multiplicity of infection (MOI) of 1,243
TCID50/cell. These experiments were performed in the presence of fetal bovine serum,
replacing pooled human serum normally used to culture these cells. Pooled human serum
inhibited PV infection of EC (data not shown) because vaccination of serum donors for
PV resulted in antibody titers that inhibited viral infection.
Immunofluorescence
EC were grown to confluence in 8-well glass chamber slides coated with
fibronectin and were infected with PV1 for the indicated times. They were then fixed
and permeabilized in ice-cold acetone for 10 min, and incubated with PV 1 antibody for
30 minutes at 37C in a humid chamber and were subsequently incubated with goat antimouse Alexa-488-labelled secondary antibody (2 g/ml) for 1 hour at room temperature.
For co-staining, PDCD8 antibody (1:100) or JunB antibody (1:500) was incubated with
EC overnight following incubation with PV1 antibody. The following day, the cells were
incubated with goat anti-mouse Alexa-488 (2 g/ml) and goat anti-rabbit Alexa-546 (2
g/ml) for 1 hour at room temperature. TO-PRO-3 iodide (1 M for 5 minutes) was used
to stain nuclei before the images were recorded by confocal microscopy.
2
Immunoblot analysis
EC infected with PV for the specific times were washed twice with PBS and then
lysed in 1x Laemmli buffer (125mmol/L Tris-HCl, pH 6.8, 1% SDS, 5% glycerol, 0.5%
ß-mercaptoethanol, 0.005% bromophenol blue).
Lysates from 500,000 cells were
resolved in an 8% (for eIF4G) or 10% (for JunB) acrylamide gel by electrophoresis and
transferred to polyvinylidene difluoride membranes and the membranes were then
blocked 1 hour at room temperature with TBST + 5% milk. The membranes were then
incubated with phospho-eIF4GI or eIF4GII (1:1000 in 5% bovine serum albumin) or
JunB (1:500) overnight at 4C. The membranes were further reacted with appropriate
horseradish peroxidase conjugated secondary antibodies. Detection was accomplished by
chemiluminescence (Amersham Biosciences) according to the manufacturer's directions.
Polysome profiling
EC were infected with PV for 4 hours as described above. Cycloheximide was
added to a final concentration of 100 ng/mL for 5 minutes and cells were then rinsed with
cold HBSS containing cycloheximide (100 ng/mL) and scraped from the dish and
pelleted by centrifugation (1000 x g, 5 minutes). The supernatant was removed and cells
were carefully resuspended in 375 L Low Salt Buffer (LSB; 20 mmol/L Tris, 10
mmol/L NaCl, 3 mmol/L MgCl2, pH 7.4) + RNasin (40 U/mL) + DTT (10 nmol/L) for 3
minutes on ice. 125 L LSB Lysis buffer (LSB containing 200 mmol/L sucrose and
1.2% Triton-X100) was then added and cells disrupted by vigorous pipetting. The
mixture was then transferred to 1.7 mL microfuge tubes and centrifuged (20,000 x g, 1
minute, 4 C) to remove nuclei and cellular debris. The supernatant was transferred to a
3
new tube containing 50 L LSB + RNasin (40 U/mL) + DTT (10 nmol/L) and 15 L
5mol/L NaCl. This mixture was then layered onto 15-50% sucrose gradients in LSB.
Gradients were centrifuged at 268,438 x g for 60 minutes (4 C) and then separated on a
density gradient flow cell fractionator (Isco Instrumentation, Lincoln, NE) coupled to a
spectrophotometer (254 nmol/L) to obtain ribosomal profiles.
Ribosomal fractions
corresponding to subpolysomes and polysomes were collected into Trizol LS (Invitrogen,
Carlsbad, CA) and RNA was isolated according to the directions of the manufacturer.
[35S]-methionine incorporation experiment
EC infected with mock media or PV for 4 hours were washed twice with DMEM
medium lacking methionine and cysteine and then incubated with methionine-free
DMEM media containing 100 Ci/ml of 35S methionine for 10 minutes at 37C. The
medium was discarded and the cells were washed twice with ice-cold PBS. The cells
were then scraped and lysed in ice cold lysis buffer (150mmol/L NaCl, 5mmol/L EDTA,
50mmol/L NaF, 1% Triton X-100, 10mmol/L Tris-HCl, 1mmol/L PMSF, 10nmol/L
NaOVa, Leupeptin 10ng/ml, Aprotinin 10ng/ml). To analyze new protein synthesis, 10
g of lysate was resolved in a 12% acrylamide gel by electrophoresis. The gel was then
mounted on Whatman filter paper, dried on gel dryer at 72C for 2 hours, and then
exposed to film for 72 hours.
Microarray hybridization
The Agilent Two-Color Low RNA Input Linear Amplification Kit is used to
generate fluorescently labeled cRNA for two-color microarray hybridizations. Agilent
4
RNA spike-in controls are combined with input total RNA samples (50 to 500 ng). The
polyadenylated fraction of the RNA sample is primed with oligo dT/T7 RNA polymerase
promoter oligonucleotide sequences and cDNA synthesis is accomplished through the
addition of MMLV-RT. Following cDNA synthesis, T7 RNA polymerase and dyelabeled nucleotides are combined with the reaction mixture to simultaneously amplify the
target material through the generation of cRNA and incorporate either cyanine 3-CTP or
cyanine 5-CTP. Fluorescently labeled, cRNA molecules are purified from the reaction
mixture using the Qiagen RNeasy mini kit. The concentration of the purified samples is
determined using a NanoDrop ND-1000 spectrophotometer.
Biotin- labeled cRNA samples (15 µg) were fragmented and combined with
Affymetrix hybridization reagents (50 pmol/L Control Oligonucleotide B2, Eukaryotic
hybridization controls, 0.1 mg/ml Herring Sperm DNA, 0.5 mg/ml BSA, 1X
Hybridization Buffer, 10% DMSO). The hybridization mixture was injected into a
GeneChip cartridge and hybridizations were performed in an Affymetrix GeneChip
Hybridization Oven 450, set to 45ºC. GeneChip cartridges were rotated within the
hybridization oven at 60 rpm for approximately 16 hours. Following hybridization, the
mixture was removed from the GeneChip cartridge and replaced with Non-Stringent
Wash Buffer (6X SSPE, 0.01% Tween-20). Wash and Stain steps on the hybridized
microarray were accomplished by loading the GeneChip into an Affymetrix Fluidics
Station 450 and running the EukGE-WS2v5_450 fluidics script.
Microarray slides were scanned in an Agilent Technologies G2505B Microarray
Scanner at 5 µmol/L resolution. The scanner performs simultaneous detection of
Cyanine-3 and Cyanine-5 signal on the hybridized slide. The scanner is capable of
5
simultaneously detecting Cyanine-3 and Cyanine-5 signal on the hybridized slide. An
extended dynamic range scan of the microarray slide is accomplished by performing a
primary scan at 100% laser power and a secondary scan at 10% power, thus generating
two separate TIF images from the scans. Pixel intensities for non-saturating features are
calculated from the primary scan. In contrast, saturating features in the primary scan
make use of the secondary scan for defining pixel intensities. In this scenario, average
pixel intensities of these features are multiplied by the magnitude of decrease in laser
power to calculate an equivalent signal representative of 100% power.
TIF files generated from the scanned microarray image are loaded into Agilent
Feature Extraction Software version 9.5.1. The software automatically positions a grid
and finds the centroid positions of each feature on the microarray. This information is
used to perform calculations that include feature intensities, background measurements
and statistical analyses. Data generated by the software is recorded as a tab-delimited
text file.
Quantitative real-time RT-PCR (qRT-PCR)
Polysome RNA was reverse transcribed using MMLV-RT (Promega, Madison,
WI) in 50 l reaction mixture. Each qRT-PCR contained 2 l cDNA and 1 l Green
Supermix reagent (Quantas). qRT-PCR was performed using an iCycler Real-Time PCR
Detection System (Bio-Rad, Hercules, CA) with denaturation step at 94C for 5 minutes,
40 cycles of 94C for 30 seconds, 57C for 30 seconds, 72C for 45 seconds and a final
elongation step of 72C for 10 minutes. Each qRT-PCR reaction was performed in
duplicate and relative differences in mRNA abundance were quantified by the
6
comparative cycle threshold (2-Ct) method 1. The primers used in this study were listed
in table 1 in supplementary data.
Construction of dicistronic vectors
The dual luciferase construct (pRF) was a gift from Dr. Elliot Spencer. To
generate the dicistronic constructs containing the 5’UTRs of candidate genes, genespecific primers were designed to cover the entire length of 5’UTRs of genes of interest
as reported in the PubMed database: PDCD8 (NM_004208), JunB (AY751746),
AMOTL2 (NM_016201), -actin (NM_001101).
Each pair of primers contained an
EcoRI site at the 5’ end and a SpeI site at the 3’ end. The 5’-UTRs of PDCD8, JunB,
AMOTL2, and -actin were first amplified in PCR reactions using HUVEC cDNA and
then were cloned into pCR2.1-TOPO according to the manufacturer’s instructions
(Invitrogen, Carlsbad, CA). Each 5’UTR was then digested out and inserted into the
dicistronic vector pRF using EcoRI and SpeI sites. The orientations and sequences of the
inserts were confirmed by DNA sequencing at the University of Utah core facility.
Transient transfections and luciferase reporter assays
EaHy 926 cells are a transformed human umbilical vein endothelial cell line2.
This cell line was created by fusing HUVECs with the human cell line A549, and they
show characteristics and functional responses similar to those of HUVECs3.
EaHy 926 cells were plated into 6-well plates 2 days prior to transfection and
allowed to reach 70-80% confluence (about 1.25 x 10 (5) cells per 6-well plate).
Lipofectamine 2000 was used for transient transfections as recommended by the
7
manufacturer (Invitrogen, Carlsbad, CA). Briefly, 5 g plasmid DNA and 5 l
lipofectamine reagent (Invitrogen, Carlsbad, CA) were mixed and transfected into EaHy
cells in separate wells in 6-well culture plates. The cells were lysed 22-24 hours posttransfection and the luciferase activity was determined using a Dual-luciferase Reporter
Assay System (Promega, Madison, WI). The assay was performed on a Synergy HT
Luminometer (Bio-TEK Instruments, Winooski, VT) using 2-second pre-measurement
delay followed by a 10-second measurement period. The results are reported as the ratio
of second cistron Firefly luciferase (fLUC) to first cistron Renilla (rLUC) luciferase
activity. The ratio of luciferase activities obtained from transfection experiments with the
control pRF plasmid was set to one. Assays were performed in duplicate and results
represent the averages of three independent experiments.
Northern blot
To obtain the Northern probe, fLUC PCR product was first cloned into pGEM-T
Easy vector by using the forward primer 5’-agagatacgccctggttcct-3’ and reverse primer
5’-cgcagtatccggaatgattt-3’. The construct was then completely linearized with NcoI
(Promega, Madison WI) and anti-sense probes were generated using SP6 transcription kit
(Promega, Madison WI). Filters with approximately 1 g polyA mRNA isolated from
transiently transfected EaHy were hybridized with [ - P]dCTP labeled fLUC specific
probes. Hybridization was carried out overnight at 60˚ C.
SUPPLEMENT REFERENCE
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Livak KJ, Schmittgen TD. Analysis of relative gene expression data using realtime quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods.
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factor VIII-related antigen established by hybridization. Proc Natl Acad Sci U S
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Kaneider NC, Leger AJ, Agarwal A, Nguyen N, Perides G, Derian C, Covic L,
Kuliopulos A. 'Role reversal' for the receptor PAR1 in sepsis-induced vascular
damage. Nat Immunol. 2007;8:1303-1312.
9
Supplement Table 1.
Molecular Function: Binding
Gene Name
Growth arrest and DNA-damage-inducible, beta
Growth arrest and DNA-damage-inducible, beta
Ephrin-A1
Nuclear receptor subfamily 4, group A, member 1
Kruppel-like factor 10
Nuclear receptor subfamily 4, group A, member 3
Ephrin-A1
Chemokine (C-X-C motif) ligand 2
Tumor necrosis factor, alpha-induced protein 3
Cysteine-rich, angiogenic inducer, 61
Adducin 2 (beta)
Chemokine (C-X-C motif) ligand 3
Pim-1 oncogene
DEAD (Asp-Glu-Ala-Asp) box polypeptide 47
Regulator of calcineurin 1
Nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor,
alpha
Chemokine (C-X-C motif) ligand 1 (melanoma growth stimulating activity,
alpha)
Early growth response 1
Chromosome 11 open reading frame 17
Homo sapiens B-cell CLL/lymphoma 6 (zinc finger protein 51) (BCL6),
transcript variant 2, mRNA [NM_138931]
CAMP responsive element binding protein 5
SNF1-like kinase
FBJ murine osteosarcoma viral oncogene homolog B
Cysteine-rich, angiogenic inducer, 61
Colony stimulating factor 2 (granulocyte-macrophage)
Endothelin 1
Nuclear receptor subfamily 4, group A, member 2
SNF1-like kinase
Cysteine-rich, angiogenic inducer, 61
Splicing factor, arginine/serine-rich 10 (transformer 2 homolog, Drosophila)
Deleted in liver cancer 1
Thymosin-like 3
CDC-like kinase 1
ADAM metallopeptidase with thrombospondin type 1 motif, 4
Basic helix-loop-helix domain containing, class B, 2
Homo sapiens Kruppel-like factor 6 (KLF6), transcript variant 1, mRNA
[NM_001008490]
GTP binding protein overexpressed in skeletal muscle
Growth arrest and DNA-damage-inducible, alpha
Hairy and enhancer of split 1, (Drosophila)
Solute carrier family 25 (mitochondrial carrier; phosphate carrier), member 25
Nexilin (F actin binding protein)
Protein kinase N2
Hairy and enhancer of split 1, (Drosophila)
Gene Identifier
NM_015675
NM_015675
NM_004428
NM_002135
NM_005655
NM_173198
NM_004428
NM_002089
NM_006290
NM_001554
NM_017488
NM_002090
NM_002648
NM_030817
NM_004414
PR
28.03
12.28
11.4
7.51
6.78
6.66
6.07
5.79
5.02
4.51
4.38
4.35
4.32
4.25
4.25
NM_020529
3.97
NM_001511
NM_001964
NM_182901
3.9
3.83
3.82
NM_138931
NM_182898
NM_173354
NM_006732
NM_001554
NM_000758
NM_001955
NM_006186
NM_173354
NM_001554
U87836
NM_182643
NM_183049
NM_004071
NM_005099
NM_003670
3.82
3.8
3.74
3.7
3.7
3.7
3.56
3.44
3.41
3.39
3.38
3.34
3.34
3.31
3.3
3.29
NM_001008490
NM_005261
NM_001924
NM_005524
NM_001006641
NM_144573
NM_006256
NM_005524
3.22
3.22
3.18
3.04
3.03
3.01
2.99
2.97
Angiomotin like 2
B-cell CLL/lymphoma 3
Colony stimulating factor 3 (granulocyte)
Early growth response 3
Interleukin 6 (interferon, beta 2)
Zinc finger protein 36, C3H type-like 2
Zinc finger CCCH-type containing 12A
Chemokine (C-X-C motif) ligand 2
Deleted in liver cancer 1
Hairy/enhancer-of-split related with YRPW motif 1
Jumonji domain containing 1C
Mitogen-activated protein kinase 8 interacting protein 2
Matrix metallopeptidase 12 (macrophage elastase)
Caldesmon 1
Jumonji domain containing 1C
Glucose-fructose oxidoreductase domain containing 1
CDC-like kinase 1
Ras-related associated with diabetes
Mannosidase, alpha, class 2A, member 2
AHNAK nucleoprotein
Homo sapiens zinc finger protein 433 (ZNF433), mRNA [NM_152602]
CNKSR family member 3
Metallothionein 2A
A kinase (PRKA) anchor protein (yotiao) 9
Interferon regulatory factor 1
Testis derived transcript (3 LIM domains)
Utrophin
Ras-related associated with diabetes
Jun B proto-oncogene
Tropomyosin 1 (alpha)
Heparin-binding EGF-like growth factor
Myosin, heavy chain 9, non-muscle
LIM and cysteine-rich domains 1
Kruppel-like factor 7 (ubiquitous)
Chemokine (C-X-C motif) ligand 3
Schwannomin interacting protein 1
Prion protein (p27-30) (Creutzfeldt-Jakob disease, Gerstmann-StrauslerScheinker syndrome, fatal familial insomnia)
Selectin L (lymphocyte adhesion molecule 1)
Leukemia inhibitory factor (cholinergic differentiation factor)
Matrix metallopeptidase 12 (macrophage elastase)
Thymosin-like 3
Apoptosis-inducing factor, mitochondrion-associated, 1
Fibronectin 1
Transcription factor-like 5 (basic helix-loop-helix)
Polo-like kinase 3 (Drosophila)
Microtubule-actin crosslinking factor 1
Isoleucyl-tRNA synthetase 2, mitochondrial
PTPRF interacting protein, binding protein 1 (liprin beta 1)
Phorbol-12-myristate-13-acetate-induced protein 1
SRY (sex determining region Y)-box 3
NM_016201
NM_005178
NM_000759
NM_004430
NM_000600
NM_006887
NM_025079
NM_002089
NM_182643
NM_012258
NM_004241
NM_012324
NM_002426
NM_033138
NM_004241
NM_018988
NM_004071
NM_004165
NM_006122
NM_001620
NM_152602
NM_173515
NM_005953
NM_147171
NM_002198
NM_152829
AK023675
NM_004165
NM_002229
NM_000366
NM_001945
NM_002473
NM_014583
NM_003709
NM_002090
NM_014575
2.96
2.92
2.9
2.89
2.89
2.88
2.85
2.85
2.83
2.81
2.81
2.8
2.79
2.78
2.75
2.73
2.73
2.71
2.71
2.71
2.66
2.64
2.64
2.62
2.6
2.59
2.58
2.58
2.56
2.55
2.55
2.53
2.52
2.51
2.48
2.48
X82545
NM_000450
NM_002309
NM_002426
NM_183049
NM_004208
NM_054034
BC065520
NM_004073
NM_033044
NM_018060
NM_003622
NM_021127
NM_005634
2.47
2.47
2.46
2.44
2.44
2.43
2.42
2.42
2.41
2.41
2.39
2.37
2.37
2.37
BCL6 co-repressor
V-abl Abelson murine leukemia viral oncogene homolog 2 (arg, Abelson-related
gene)
Methylcrotonoyl-Coenzyme A carboxylase 1 (alpha)
Phorbol-12-myristate-13-acetate-induced protein 1
Homo sapiens CREB regulated transcription coactivator 1 (CRTC1), transcript
variant 2, mRNA [NM_025021]
PRP4 pre-mRNA processing factor 4 homolog B (yeast)
Synovial sarcoma translocation gene on chromosome 18-like 1
Zinc finger protein 784
Phorbol-12-myristate-13-acetate-induced protein 1
Hyperpolarization activated cyclic nucleotide-gated potassium channel 2
Phorbol-12-myristate-13-acetate-induced protein 1
Electron-transferring-flavoprotein dehydrogenase
Leucine-rich repeat-containing G protein-coupled receptor 4
Myosin VI
Phorbol-12-myristate-13-acetate-induced protein 1
Phorbol-12-myristate-13-acetate-induced protein 1
Dual specificity phosphatase 1
Zinc finger protein 789
GRIP and coiled-coil domain containing 2
Heparin-binding EGF-like growth factor
Connective tissue growth factor
Metallothionein 2A
Membrane-associated ring finger (C3HC4) 7
Retinitis pigmentosa 9 (autosomal dominant)
C-mer proto-oncogene tyrosine kinase
V-maf musculoaponeurotic fibrosarcoma oncogene homolog K (avian)
Chromodomain helicase DNA binding protein 2
Intestine-specific homeobox
RANBP2-like and GRIP domain containing 5
Phorbol-12-myristate-13-acetate-induced protein 1
Chemokine (C-C motif) ligand 2
Phorbol-12-myristate-13-acetate-induced protein 1
Similar to hCG2024106
Tumor necrosis factor, alpha-induced protein 3
Secreted phosphoprotein 1 (osteopontin, bone sialoprotein I, early T-lymphocyte
activation 1)
Integrin, beta 2 (complement component 3 receptor 3 and 4 subunit)
Methylmalonyl Coenzyme A mutase
Secreted phosphoprotein 1 (osteopontin, bone sialoprotein I, early T-lymphocyte
activation 1)
Filamin B, beta (actin binding protein 278)
Ras homolog gene family, member B
Heat shock protein 90kDa beta (Grp94), member 1
ADP-ribosylation factor-like 5A
Glycoprotein (transmembrane) nmb
Homo sapiens RIO kinase 3 (yeast) (RIOK3), transcript variant 2, mRNA
[NM_145906]
V-fos FBJ murine osteosarcoma viral oncogene homolog
Phorbol-12-myristate-13-acetate-induced protein 1
NM_017745
2.37
NM_007314
NM_020166
NM_021127
2.35
2.34
2.34
NM_025021
NM_003913
NM_198935
NM_203374
NM_021127
NM_001194
NM_021127
NM_004453
NM_018490
NM_004999
NM_021127
NM_021127
NM_004417
AK131429
NM_181453
NM_001945
NM_001901
NM_005953
NM_022826
NM_203288
U08023
NM_002360
BC031320
NM_001008494
NM_005054
NM_021127
NM_002982
NM_021127
NM_013440
NM_006290
2.34
2.34
2.34
2.33
2.31
2.3
2.3
2.29
2.29
2.28
2.28
2.27
2.26
2.26
2.24
2.24
2.24
2.23
2.23
2.22
2.21
2.21
2.21
2.2
2.19
2.18
2.17
2.17
2.17
2.16
NM_000582
NM_000211
NM_000255
2.16
2.16
2.16
NM_000582
NM_001457
NM_004040
NM_003299
NM_012097
NM_001005340
2.15
2.15
2.14
2.14
2.14
2.14
NM_145906
NM_005252
NM_021127
2.14
2.14
2.13
Inhibin, beta A
Insulin induced gene 1
Secreted phosphoprotein 1 (osteopontin, bone sialoprotein I, early T-lymphocyte
activation 1)
GLI-Kruppel family member GLI4
AHA1, activator of heat shock 90kDa protein ATPase homolog 2 (yeast)
Secreted phosphoprotein 1 (osteopontin, bone sialoprotein I, early T-lymphocyte
activation 1)
Inositol 1,4,5-trisphosphate 3-kinase C
Hypothetical protein KIAA1434
FOS-like antigen 2
Human immunodeficiency virus type I enhancer binding protein 1
Phorbol-12-myristate-13-acetate-induced protein 1
Serine/threonine kinase 38 like
Kruppel-like factor 2 (lung)
Interleukin 1 receptor-like 1
Platelet-derived growth factor alpha polypeptide
Zinc finger, CCHC domain containing 10
Secreted phosphoprotein 1 (osteopontin, bone sialoprotein I, early T-lymphocyte
activation 1)
Zinc finger protein 625
Snail homolog 1 (Drosophila)
RAR-related orphan receptor A
2-5-oligoadenylate synthetase-like
Secreted phosphoprotein 1 (osteopontin, bone sialoprotein I, early T-lymphocyte
activation 1)
Secreted phosphoprotein 1 (osteopontin, bone sialoprotein I, early T-lymphocyte
activation 1)
Early growth response 2 (Krox-20 homolog, Drosophila)
Tubulin tyrosine ligase
Suppressor of cytokine signaling 2
Cardiotrophin-like cytokine factor 1
Family with sequence similarity 80, member B
Von Willebrand factor
RANBP2-like and GRIP domain containing 1
Zinc finger protein 579
Runt-related transcription factor 1 (acute myeloid leukemia 1; aml1 oncogene)
BCL2-associated X protein
Pim-3 oncogene
Interleukin 8
SEC31 homolog A (S. cerevisiae)
La ribonucleoprotein domain family, member 2
Secreted phosphoprotein 1 (osteopontin, bone sialoprotein I, early T-lymphocyte
activation 1)
Split hand/foot malformation (ectrodactyly) type 1
Neuronal PAS domain protein 3
Interferon-related developmental regulator 1
LIM and calponin homology domains 1
Cyclic AMP phosphoprotein, 19 kD
Myosin VI
ADP-ribosylation factor-like 5A
Calcium channel, voltage-dependent, R type, alpha 1E subunit
NM_002192
AW993939
2.13
2.12
NM_000582
CB050471
NM_152392
2.12
2.12
2.11
NM_000582
NM_025194
NM_019593
NM_005253
NM_002114
NM_021127
NM_015000
NM_016270
NM_016232
NM_002607
NM_017665
2.11
2.11
2.11
2.1
2.1
2.1
2.1
2.09
2.09
2.08
2.08
NM_000582
NM_145233
NM_005985
NM_134260
NM_003733
2.07
2.07
2.07
2.07
2.06
NM_000582
2.06
NM_000582
NM_000399
NM_153712
NM_003877
NM_013246
NM_020734
NM_000552
NM_001024457
NM_152600
D43967
NM_138763
NM_001001852
NM_000584
AK128047
NM_032239
2.06
2.06
2.05
2.05
2.05
2.05
2.05
2.05
2.04
2.04
2.04
2.04
2.03
2.03
2.03
NM_000582
NM_006304
NM_173159
NM_001007245
NM_014988
NM_006628
NM_004999
NM_012097
NM_000721
2.02
2.02
2.02
2.02
2.02
2.01
2
2
2
Molecular Function: Catalytic activity
Gene Name
Tumor necrosis factor, alpha-induced protein 3
Pim-1 oncogene
DEAD (Asp-Glu-Ala-Asp) box polypeptide 47
SNF1-like kinase
SNF1-like kinase
Splicing factor, arginine/serine-rich 10 (transformer 2 homolog, Drosophila)
CDC-like kinase 1
ADAM metallopeptidase with thrombospondin type 1 motif, 4
Glutaminase
Protein kinase N2
Jumonji domain containing 1C
Matrix metallopeptidase 12 (macrophage elastase)
Jumonji domain containing 1C
Glucose-fructose oxidoreductase domain containing 1
CDC-like kinase 1
Ras-related associated with diabetes
Dual specificity phosphatase 5
Mannosidase, alpha, class 2A, member 2
A kinase (PRKA) anchor protein (yotiao) 9
Ras-related associated with diabetes
Myosin, heavy chain 9, non-muscle
Matrix metallopeptidase 12 (macrophage elastase)
Apoptosis-inducing factor, mitochondrion-associated, 1
Fibronectin 1
Polo-like kinase 3 (Drosophila)
Isoleucyl-tRNA synthetase 2, mitochondrial
BCL6 co-repressor
V-abl Abelson murine leukemia viral oncogene homolog 2 (arg, Abelson-related
gene)
Methylcrotonoyl-Coenzyme A carboxylase 1 (alpha)
PRP4 pre-mRNA processing factor 4 homolog B (yeast)
Electron-transferring-flavoprotein dehydrogenase
Myosin VI
Dual specificity phosphatase 1
Membrane-associated ring finger (C3HC4) 7
C-mer proto-oncogene tyrosine kinase
Chromodomain helicase DNA binding protein 2
Chemokine (C-C motif) ligand 2
Tumor necrosis factor, alpha-induced protein 3
Methylmalonyl Coenzyme A mutase
Ras homolog gene family, member B
Homo sapiens RIO kinase 3 (yeast) (RIOK3), transcript variant 2, mRNA
[NM_145906]
Tryptase gamma 1
Inositol 1,4,5-trisphosphate 3-kinase C
Hypothetical protein KIAA1434
Serine/threonine kinase 38 like
Gene Identifier
NM_006290
NM_002648
NM_030817
NM_173354
NM_173354
U87836
NM_004071
NM_005099
NM_014905
NM_006256
NM_004241
NM_002426
NM_004241
NM_018988
NM_004071
NM_004165
NM_004419
NM_006122
NM_147171
NM_004165
NM_002473
NM_002426
NM_004208
NM_054034
NM_004073
NM_018060
NM_017745
PR
5.02
4.32
4.25
3.74
3.41
3.38
3.31
3.3
3.21
2.99
2.81
2.79
2.75
2.73
2.73
2.71
2.71
2.71
2.62
2.58
2.53
2.44
2.43
2.42
2.41
2.39
2.37
NM_007314
NM_020166
NM_003913
NM_004453
NM_004999
NM_004417
NM_022826
U08023
BC031320
NM_002982
NM_006290
NM_000255
NM_004040
2.35
2.34
2.34
2.29
2.28
2.26
2.23
2.21
2.21
2.17
2.16
2.16
2.14
NM_145906
NM_012467
NM_025194
NM_019593
NM_015000
2.14
2.11
2.11
2.11
2.1
2-5-oligoadenylate synthetase-like
Tubulin tyrosine ligase
Family with sequence similarity 80, member B
Homo sapiens signal peptide peptidase-like 2B (SPPL2B), transcript variant 1,
mRNA [NM_020172]
Pim-3 oncogene
Split hand/foot malformation (ectrodactyly) type 1
Myosin VI
Histidine triad nucleotide binding protein 3
NM_003733
NM_153712
NM_020734
2.06
2.05
2.05
NM_020172
NM_001001852
NM_006304
NM_004999
NM_138571
2.04
2.04
2.02
2
2
Gene Identifier
NM_002135
NM_005655
NM_173198
NM_004414
NM_001964
PR
7.51
6.78
6.66
4.25
3.83
NM_138931
NM_182898
NM_006732
NM_006186
NM_003670
3.82
3.8
3.7
3.44
3.29
NM_001008490
NM_005524
NM_005524
NM_005178
NM_004430
NM_006887
NM_012258
NM_005966
NM_002198
NM_002229
NM_014583
NM_003709
BC065520
NM_017745
3.22
3.04
2.97
2.92
2.89
2.88
2.81
2.74
2.6
2.56
2.52
2.51
2.42
2.37
NM_025021
NM_002360
NM_001008494
NM_005252
NM_005253
NM_016270
NM_134260
NM_000399
D43967
NM_173159
2.34
2.21
2.2
2.14
2.1
2.09
2.07
2.06
2.04
2.02
Molecular Function: Transcription regulator activity
Gene Name
Nuclear receptor subfamily 4, group A, member 1
Kruppel-like factor 10
Nuclear receptor subfamily 4, group A, member 3
Regulator of calcineurin 1
Early growth response 1
Homo sapiens B-cell CLL/lymphoma 6 (zinc finger protein 51) (BCL6),
transcript variant 2, mRNA [NM_138931]
CAMP responsive element binding protein 5
FBJ murine osteosarcoma viral oncogene homolog B
Nuclear receptor subfamily 4, group A, member 2
Basic helix-loop-helix domain containing, class B, 2
Homo sapiens Kruppel-like factor 6 (KLF6), transcript variant 1, mRNA
[NM_001008490]
Hairy and enhancer of split 1, (Drosophila)
Hairy and enhancer of split 1, (Drosophila)
B-cell CLL/lymphoma 3
Early growth response 3
Zinc finger protein 36, C3H type-like 2
Hairy/enhancer-of-split related with YRPW motif 1
NGFI-A binding protein 1 (EGR1 binding protein 1)
Interferon regulatory factor 1
Jun B proto-oncogene
LIM and cysteine-rich domains 1
Kruppel-like factor 7 (ubiquitous)
Transcription factor-like 5 (basic helix-loop-helix)
BCL6 co-repressor
Homo sapiens CREB regulated transcription coactivator 1 (CRTC1), transcript
variant 2, mRNA [NM_025021]
V-maf musculoaponeurotic fibrosarcoma oncogene homolog K (avian)
Intestine-specific homeobox
V-fos FBJ murine osteosarcoma viral oncogene homolog
FOS-like antigen 2
Kruppel-like factor 2 (lung)
RAR-related orphan receptor A
Early growth response 2 (Krox-20 homolog, Drosophila)
Runt-related transcription factor 1 (acute myeloid leukemia 1; aml1 oncogene)
Neuronal PAS domain protein 3
Molecular Function: Molecular transducer activity
Gene Name
Nuclear receptor subfamily 4, group A, member 1
Nuclear receptor subfamily 4, group A, member 3
Nuclear receptor subfamily 4, group A, member 2
Opioid growth factor receptor-like 1
CD274 molecule
Heparin-binding EGF-like growth factor
Polo-like kinase 3 (Drosophila)
G protein-coupled receptor 78
Leucine-rich repeat-containing G protein-coupled receptor 4
Pyrimidinergic receptor P2Y, G-protein coupled, 4
Heparin-binding EGF-like growth factor
C-mer proto-oncogene tyrosine kinase
Chemokine (C-C motif) ligand 2
Similar to hCG2024106
Integrin, beta 2 (complement component 3 receptor 3 and 4 subunit)
Inhibin, beta A
Interleukin 1 receptor-like 1
RAR-related orphan receptor A
Suppressor of cytokine signaling 2
Neuronal PAS domain protein 3
Gene Identifier
NM_002135
NM_173198
NM_006186
NM_024576
NM_014143
NM_001945
NM_004073
NM_080819
NM_018490
NM_002565
NM_001945
U08023
NM_002982
NM_013440
NM_000211
NM_002192
NM_016232
NM_134260
NM_003877
NM_173159
PR
7.51
6.66
3.44
3.25
2.91
2.55
2.41
2.31
2.29
2.26
2.24
2.21
2.17
2.17
2.16
2.13
2.09
2.07
2.05
2.02
Gene Identifier
PR
Molecular Function: Enzyme regulator activity
Gene Name
Chemokine (C-X-C motif) ligand 1 (melanoma growth stimulating activity,
alpha)
Deleted in liver cancer 1
Deleted in liver cancer 1
Mitogen-activated protein kinase 8 interacting protein 2
AHA1, activator of heat shock 90kDa protein ATPase homolog 2 (yeast)
NM_001511
NM_182643
NM_182643
NM_012324
NM_152392
3.9
3.34
2.83
2.8
2.11
Gene Identifier
NM_018988
NM_004208
NM_004453
PR
2.73
2.43
2.29
Gene Identifier
NM_012324
NM_000366
NM_054034
PR
2.8
2.55
2.42
Gene Identifier
NM_001194
NM_000721
PR
2.3
2
Electron carrier activity
Gene Name
Glucose-fructose oxidoreductase domain containing 1
Apoptosis-inducing factor, mitochondrion-associated, 1
Electron-transferring-flavoprotein dehydrogenase
Molecular Function: Structural molecule activity
Gene Name
Mitogen-activated protein kinase 8 interacting protein 2
Tropomyosin 1 (alpha)
Fibronectin 1
Molecular Function: Transporter activity
Gene Name
Hyperpolarization activated cyclic nucleotide-gated potassium channel 2
Calcium channel, voltage-dependent, R type, alpha 1E subunit
Molecular Function: Auxiliary transport protein activity
Gene Name
Cyclic AMP phosphoprotein, 19 kD
Gene Identifier
NM_006628
PR
2.01
Gene Identifier
NM_152392
PR
2.11
Molecular Function: Chaperone regulator activity
Gene Name
AHA1, activator of heat shock 90kDa protein ATPase homolog 2 (yeast)
Supplement Table 2.
Gene Name Description
MYH9
FLNB
PDCD8
AHNAK
MYO6
PILRB
CMIP
TPSG1
HSP90B1
C6orf111
PDGFA
PKN2
AY029066
AMOTL2
CYR61
PIM1
TNFAIP3
JunB
EFNA1
GADD45B
Homo sapiens myosin, heavy polypeptide 9, non-muscle (MYH9), mRNA
[NM 002473]
Homo sapiens filamin B, beta (actin binding protein 278) (FLNB), mRNA
[NM 001457]
Homo sapiens programmed cell death 8 (apoptosis-inducing factor)
(PDCD8), nuclear gene encoding mitochondrial protein, transcript variant 1,
mRNA [NM 004208]
Homo sapiens AHNAK nucleoprotein (desmoyokin) (AHNAK), transcript
variant 1, mRNA [NM 001620]
Homo sapiens myosin VI (MYO6), mRNA [NM_004999]
Homo sapiens paired immunoglobin-like type 2 receptor beta (PILRB),
transcript variant 1, mRNA [NM 013440]
Homo sapiens c-Maf-inducing protein (CMIP), transcript variant C-mip,
mRNA [NM 198390]
Homo sapiens tryptase gamma 1 (TPSG1), mRNA [NM_012467]
Homo sapiens heat shock protein 90kDa beta (Grp94), member 1
(HSP90B1), mRNA [NM 003299]
Homo sapiens chromosome 6 open reading frame 111 (C6orf111), mRNA
[NM 032870]
Homo sapiens platelet-derived growth factor alpha polypeptide (PDGFA),
transcript variant 1, mRNA [NM 002607]
Homo sapiens protein kinase N2 (PKN2), mRNA [NM_006256]
Homo sapiens Humanin (HN1) mRNA, complete cds. [AY029066]
Homo sapiens angiomotin like 2 (AMOTL2), mRNA [NM_016201]
Homo sapiens cysteine-rich, angiogenic inducer, 61 (CYR61), mRNA
[NM 001554]
Homo sapiens pim-1 oncogene (PIM1), mRNA [NM_002648]
Homo sapiens tumor necrosis factor, alpha-induced protein 3 (TNFAIP3),
mRNA [NM 006290]
Homo sapiens jun B proto-oncogene (JUNB), mRNA [NM_002229]
Homo sapiens ephrin-A1 (EFNA1), transcript variant 1, mRNA
[NM 004428]
Homo sapiens growth arrest and DNA-damage-inducible, beta (GADD45B),
mRNA [NM 015675]
Forward primer
sequences
gaagagctagaggcgctgaa
Reverse primer
sequences
ctttgccttctcgaggtttg
PCR product
size
243bp
cgtgatggtgtttgttgagg
gatgtgctgtcctgcaaaga
151bp
tcttccccgagaaaggaaat
aactcaacattgggctccag
220bp
tgagctggagtgtcctgatg
acttgggccctttcaacttt
242bp
aggctgaggcgtattcaaga
gggcagtccttctacagcac
ttgggattcctcctccttct
gctcgactcggcagaaatac
222bp
154bp
ccagtttgcttcaacccatt
gtaacaggagcccatgagga
245bp
cctgcctacgtgaactggat
tgggaagaggttccagaatg
acttggattcctgccatcag
gttgccagaccatccgtact
208bp
220bp
tcatcattcagggatcgtca
atcccctccttcagcatctt
228bp
acgtcaggaagaagccaaaa
ggctcatcctcacctcacat
200bp
ttccggctaattgattggag
aaatcttaccccgcctgttt
gcttcaatgagggtctgctc
ctccctgtttttggaatgga
tgtgagtcagcttggtttcg
ggcaggtcaatttcactggt
gaaaacagatggcaccgact
tggtcttgctgcatttcttg
244bp
209bp
188bp
209bp
gcaaatagcagcctttctgg
gagagcacaatggctgaaca
cctaggacccctggagagtc
tccagtgtgtatcggtgcat
363bp
155bp
tggaacagcccttctaccac
gagacagtcctttcccacca
gaagaggcgagcttgagaga
ctggcttccaagcaagaaac
241bp
208bp
aatccacttcacgctcatcc
gaccaggagacaatgcaggt
151bp
ARMCX2
FN1
THBS1
LAMB1
PPAP2B
CYP26B1
TBCC
CDC42EP2
NR3C1
EIF1AX
ACTB
Homo sapiens armadillo repeat containing, X-linked 2 (ARMCX2), mRNA
[NM 014782]
Homo sapiens fibronectin 1 (FN1), transcript variant 1, mRNA
[NM 212482]
Homo sapiens thrombospondin 1 (THBS1), mRNA [NM_003246]
Homo sapiens laminin, beta 1 (LAMB1), mRNA [NM_002291]
Homo sapiens phosphatidic acid phosphatase type 2B (PPAP2B), transcript
variant 1, mRNA [NM 003713]
Homo sapiens cytochrome P450, family 26, subfamily B, polypeptide 1
(CYP26B1), mRNA [NM 019885]
Homo sapiens tubulin-specific chaperone c (TBCC), mRNA [NM_003192]
Homo sapiens CDC42 effector protein (Rho GTPase binding) 2
(CDC42EP2), mRNA [NM 006779]
Homo sapiens nuclear receptor subfamily 3, group C, member 1
(glucocorticoid receptor) (NR3C1), transcript variant 5, mRNA
[NM 000176]
Homo sapiens eukaryotic translation initiation factor 1A, X-linked
(EIF1AX), mRNA [NM 001412]
Homo sapiens actin, beta (ACTB), mRNA [NM_001101]
tctgctctggacacagttgg
tattgcagaagccattgcag
156bp
accaacctacggatgactcg
gctcatcatctggccatttt
230bp
ttgtctttggaaccacacca
aacgtggttggaagaacctg
tcgagacaagcaccatcaag
ctggacagctcatcacagga
acactccctggaaacagtgg
accgcgacttcttcaggtaa
187bp
151bp
186bp
acacggtgtccaattccatt
gcctcctggtacacgttgat
172bp
gttgaaaggcggaaacaaaa
tcgtaagcggctaggaaaaa
209bp
gatcaggacctggacagcat
aacacggctcagaaggagaa
177bp
taccctgcatgtacgaccaa
tccttccctcttgacaatgg
212bp
gcattgctgcttttcctacc
ttctgcctccctcaaattgt
177bp
tggagaagagctatgagctgcctg
gtgccaccagacagcactgtgttg
202bp
Supplement Table 3.
Gene Name
GADD45B
EFNA1
Description
Homo sapiens growth arrest and DNA-damage-inducible, beta (GADD45B), mRNA
[NM_015675]
Array
PR
qRTPCR PR
LogArray
PR
Logq
RT-PCR PR
28
13.67
1.45
1.14
11.4
9.65
1.06
0.98
TNFAIP3
Homo sapiens ephrin-A1 (EFNA1), transcript variant 1, mRNA [NM_004428]
Homo sapiens tumor necrosis factor, alpha-induced protein 3 (TNFAIP3), mRNA
[NM_006290]
5.02
3.24
0.70
0.51
PIM1
Homo sapiens pim-1 oncogene (PIM1), mRNA [NM_002648]
4.32
6.14
0.64
0.79
CYR61
3.39
6.32
0.53
0.80
C6orf111
Homo sapiens cysteine-rich, angiogenic inducer, 61 (CYR61), mRNA [NM_001554]
Homo sapiens chromosome 6 open reading frame 111 (C6orf111), mRNA
[NM_032870]
3.13
3.34
0.50
0.52
PKN2
Homo sapiens protein kinase N2 (PKN2), mRNA [NM_006256]
2.99
1.34
0.48
0.13
AMOTL2
Homo sapiens angiomotin like 2 (AMOTL2), mRNA [NM_016201]
Homo sapiens AHNAK nucleoprotein (desmoyokin) (AHNAK), transcript variant 1,
mRNA [NM_001620]
Homo sapiens myosin, heavy polypeptide 9, non-muscle (MYH9), mRNA
[NM_002473]
2.96
2.41
0.47
0.38
2.71
3.06
0.43
0.49
2.53
1.847
0.40
0.27
2.48
1.91
0.39
0.28
2.43
1.8
0.39
0.26
2.17
2.65
0.34
0.42
2.15
2.23
0.33
0.35
2.14
1.42
0.33
0.15
2.11
1.72
0.32
0.24
2.08
0.8
0.32
-0.10
2.02
0.64
0.31
-0.19
AHNAK
MYH9
AY029066
PDCD8
PILRB
FLNB
HSP90B1
TPSG1
Homo sapiens Humanin (HN1) mRNA, complete cds. [AY029066]
Homo sapiens programmed cell death 8 (apoptosis-inducing factor) (PDCD8),
nuclear gene encoding mitochondrial protein, transcript variant 1, mRNA
[NM_004208]
Homo sapiens paired immunoglobin-like type 2 receptor beta (PILRB), transcript
variant 1, mRNA [NM_013440]
Homo sapiens filamin B, beta (actin binding protein 278) (FLNB), mRNA
[NM_001457]
Homo sapiens heat shock protein 90kDa beta (Grp94), member 1 (HSP90B1), mRNA
[NM_003299]
CMIP
Homo sapiens tryptase gamma 1 (TPSG1), mRNA [NM_012467]
Homo sapiens platelet-derived growth factor alpha polypeptide (PDGFA), transcript
variant 1, mRNA [NM_002607]
Homo sapiens c-Maf-inducing protein (CMIP), transcript variant C-mip, mRNA
[NM_198390]
MYO6
Homo sapiens myosin VI (MYO6), mRNA [NM_004999]
2
1.65
0.30
0.22
JunB
Homo sapiens jun B proto-oncogene (JUNB), mRNA [NM_002229]
2
0.89
0.30
-0.05
PDGFA
FN1
Homo sapiens fibronectin 1 (FN1), transcript variant 1, mRNA [NM_212482]
1.64
2.64
0.21
0.42
LAMB1
Homo sapiens laminin, beta 1 (LAMB1), mRNA [NM_002291]
1.24
0.7
0.09
-0.15
THBS1
Homo sapiens thrombospondin 1 (THBS1), mRNA [NM_003246]
Homo sapiens armadillo repeat containing, X-linked 2 (ARMCX2), mRNA
[NM_014782]
Homo sapiens phosphatidic acid phosphatase type 2B (PPAP2B), transcript variant 1,
mRNA [NM_003713]
1.18
1.16
0.07
0.06
1.03
0.84
0.01
-0.08
0.87
1.1
-0.06
0.04
0.59
0.44
-0.23
-0.36
0.42
0.49
-0.38
-0.31
0.42
0.21
-0.38
-0.68
0.4
0.71
-0.40
-0.15
0.25
0.17
-0.60
-0.77
0.14
0.07
-0.85
-1.15
ARMCX2
PPAP2B
ACTB
EIF1AX
NR3C1
CDC42EP2
TBCC
CYP26B1
Homo sapiens actin, beta (ACTB), mRNA [NM_001101]
Homo sapiens eukaryotic translation initiation factor 1A, X-linked (EIF1AX), mRNA
[NM_001412]
Homo sapiens nuclear receptor subfamily 3, group C, member 1 (glucocorticoid
receptor) (NR3C1), transcript variant 5, mRNA [NM_000176]
Homo sapiens CDC42 effector protein (Rho GTPase binding) 2 (CDC42EP2),
mRNA [NM_006779]
Homo sapiens tubulin-specific chaperone c (TBCC), mRNA [NM_003192]
Homo sapiens cytochrome P450, family 26, subfamily B, polypeptide 1 (CYP26B1),
mRNA [NM_019885]